Glycosylation is a common post-translational modification of proteins. Glycans are implicated in a wide range of biological events such as cell-cell interactions and recognition, inflammation, and autoimmune diseases (Ohtsubo and Marth Cell 126:855-867 (2006); Varki Glycobiology 3:97-130 (1993)). Detailed knowledge of glycan structures would facilitate structure-function correlations. This can be achieved by developing tools for highly sensitive analysis of glycan chains. For example, structural analysis of asparagine-linked carbohydrates (N-linked glycans) can be performed by releasing sugars from the protein backbone using enzymes such as PNGase F (Tarentino and Plummer Methods Enzymol. 230:44-57 (1994)). The O-linked glycans are most commonly attached to serine or threonine residues through the GaINAc residue at the reducing end. So far there is no enzymatic way of releasing of O-glycans intact. Consequently, this is achieved by chemical methods, typically by β-elimination with mild alkali (Kakehi et al. J Chromatogr A. 680:209-215 (1994)) or mild hydrazinolysis (Royle et al. Anal Biochem. 304:70-90 (2002)). O-linked disaccharides (Core 1 type O-glycan) are among the most abundant core structures found in mucin glycoproteins, for example, the Thomsen-Freidenreich antigen (T antigen) immunodeterminant group and is used as a specific marker of carcinoma (Ohtsubo and Marth Cell 126:855-867 (2006); Varki Glycobiology 3:97-130 (1993)).
To date, endo-α-GalNAcases have been purified from Clostridium perfringens (Huang and Aminoff J Biol Chem. 247:6737-6742 (1972)); Streptococcus pneumoniae (Umemoto et al. J Biol Chem. 252:8609-8614 (1977), Glasgow et al. J Biol Chem. 252:8615-8623 (1977) and Brooks and Savage Glycoconj J. 14:183-190 (1997)); Alcaligenes sp. (Fan et al. Agric Biol Chem. 54:233-234 (1990)); Bacillus sp. (Ashida et al. Arch Biochem Biophys. 373:394-400 (2000)); Bifidobacterium longum (Fujita et al. J Biol Chem. 280:37415-37422 (2005) and U.S. Publication No. 2006/0223140); and Streptomyces (Ishii-Karakasa et al. Biochem J. 288:475-482 (1992) and Ishii-Karakasa et al. Eur J Biochem. 247:709-715 (1997)). All of the enzymes have a narrow substrate specificity, acting only on the α-linked disaccharide, Galβ1,3GalNAc.